In the analysis of biological materials, it is often necessary to first isolate or purify molecules of interest from a mixture of molecules such as found in a biological sample. While different molecules can be separated based on their chemical properties, such separation methods can be tedious and time consuming. In the analysis of nucleic acid molecules, if the desired molecules are not sufficient to be detected easily, techniques can be used to increase their copy numbers for easy detection.
For example, DNA analysis and sequencing typically involves increasing the copy number of a targeted portion of the genome by PCR amplification. Cleanup of PCR products to remove primers and dNTP is necessary prior to sequencing because residual primers can act as extension primers during sequencing and produce dye-labeled sequence fragments that can complicate data interpretation or, worse, lead to false conclusions. Further, failure to remove dNTP can result in suboptimal ratios of dNTP to labeled ddNTP (di-deoxynucleotides) used to regulate read lengths in sequencing reactions. Similar considerations apply to removal of unextended primers from samples prior to MALDI-TOF analysis.
Conventional approaches to PCR cleanup generally rely on immobilization of DNA amplicons, primers and dNTP on solid supports (e.g., membranes or magnetic particles), typically using chaotropic salts, and then selectively eluting undesired components off these surfaces in various solvents until the desired component is eluted. These multiple step processes are time-consuming, labor intensive and introduce undesirable components such as organic solvents and chaotropic salts that interfere with subsequent reactions and therefore require further cleanup.
In order to improve usability of polymerase chain reaction (PCR) amplicons for downstream applications, it is desirable to remove excess primers and unincorporated deoxynucleotide triphosphates (dNTPs) in a more efficient manner while retaining the amplified double-stranded (ds) DNA (nucleic acids).